RESEARCH ARTICLE
Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4 Anantha R. Nookala1☯, Junhao Li2☯, Anusha Ande1, Lei Wang2, Naveen K. Vaidya3, Weihua Li2, Santosh Kumar4, Anil Kumar1* 1 Division of Pharmacology & Toxicology, School of Pharmacy, University of Missouri Kansas City, Kansas City, Missouri, United States of America, 2 Shanghai key laboratory of new drug design, School of Pharmacy, East China University of Science and Technology, Shanghai, China, 3 Department of Mathematics and Statistics, University of Missouri Kansas City, Kansas City, Missouri, United States of America, 4 Department of Pharmaceutical Sciences, College of pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America ☯ These authors contributed equally to this work. *
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OPEN ACCESS Citation: Nookala AR, Li J, Ande A, Wang L, Vaidya NK, Li W, et al. (2016) Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4. PLoS ONE 11(1): e0146529. doi:10.1371/journal.pone.0146529 Editor: Sanjay B. Maggirwar, University of Rochester Medical Center, UNITED STATES Received: November 20, 2015 Accepted: December 19, 2015 Published: January 7, 2016 Copyright: © 2016 Nookala et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The work was supported by grants (DA025011 and DA 025528) from NIDA/NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: AK is an editorial board member, but this does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.
Abstract Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially
PLOS ONE | DOI:10.1371/journal.pone.0146529 January 7, 2016
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with ritonavir-boosted antiretroviral therapy, in HIV-1-infected individuals who abuse methamphetamine.
Introduction Cytochrome P450 (CYP) belongs to the family of heme proteins that are involved in the biotransformation of xenobiotics. Among the various CYP isozymes, CYP3A4 metabolizes approximately 50% of the currently marketed drugs including several antiretroviral drugs [1]. Most antiretroviral drugs including protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) are substrates, inducers and/or inhibitors of CYP3A4 [2]. The highly active antiretroviral therapy (HAART) regimens contain multiple drugs including PIs and NNRTIs, which also causes potential drug-drug interactions through CYP3A4 [3, 4]. Among all the PIs, ritonavir is known to be the strongest inhibitor of CYP3A4 and is, therefore, used as a booster in the HAART regimen that contains NNRTIs and PIs. Based on the physicochemical properties of the PIs, several studies have shown that PIs exhibit two types of spectral changes upon binding with CYP3A4 known as type I and type II [5, 6]. Methamphetamine is a commonly used substance of abuse among HIV-1 infected population. Studies have shown that the risk of acquiring HIV-1 infection is higher among men who have sex with men (MSM) and abuse amphetamines compared to those MSM who don’t abuse drugs [7, 8]. Furthermore, several in vitro and in vivo studies have shown the role of methamphetamine in altering HIV-1 pathogenesis by various mechanisms, including suppression of innate restriction factors in macrophages and increased viral loads in the brain [9, 10]. Few reports have shown the occurrence of fatal interactions in patients abusing methamphetamine while on ritonavir therapy resulting from inhibition of CYP2D6 mediated methamphetamine metabolism [11]. However, it’s unclear whether methamphetamine also reduces the response to HAART by altering the bioavailability of HAART and increasing HAART-mediated toxicity that would ultimately result in increased HIV-1 pathogenesis in methamphetamine users. Since, methamphetamine is also partly metabolized by CYP3A4, which is known to metabolize NNRTIs and PIs, we propose that methamphetamine interacts with NNRTIs/PIs through CYP3A4. Therefore, in this study we determined the interaction of methamphetamine with CYP3A4 followed by the effect of methamphetamine on the interaction of PIs with CYP3A4 using spectral binding and docking studies.
Materials and Methods Materials Plasmid encoding CYP3A4 was generously provided by Dr. James Halpert (Skaggs School of pharmacy and pharmaceutical sciences, UCSD). Ni-NTA agarose column was obtained from Qiagen (Valencia, CA). Methamphetamine was purchased from Sigma chemicals (St.Louis, MO, USA). All the protease inhibitors (PIs) were obtained from NIH AIDS reagent center. XTerra 1 MS C18 column (4.6X50mm, i.d, 3.5 μm) was purchased from Waters (Milford, MA, USA). The other reagents used in the study were obtained from standard commercial sources.
Enzyme preparation Histidine tagged CYP3A4 enzyme was expressed in E. coli and purified on Ni-NTA agarose column as described previously [12]. Briefly, CYP3A4 cDNA was introduced into TOPP3
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strain of E. coli by transformation and the cells were plated on LB ampicillin plate with tetracycline. The next day, a viable and isolated colony was grown in TB media for 48 h to induce CYP3A4. The cultures were harvested by centrifugation and CYP3A4 was extracted from the membrane using detergent followed by ultracentrifugation. The supernatant was loaded on a Ni-NTA agarose column for further purification. The purified enzyme was dialyzed and final preparation was stored at -80οC until use.
Spectral binding assay The spectral change was recorded by using a UV/Vis double beam spectrophotometer (6800 UV/VIS JENWAY spectrophotometer) as described earlier [6]. Briefly, 0.5 μM of purified CYP3A4 enzyme was incubated with 2 ml of 0.1 M HEPES buffer for 3 min and distributed equally between the sample and reference cuvettes. Depending upon the solvent used to solubilize methamphetamine or PIs, equal quantities of either methanol or water was added into the reference cuvette with final volume of the external solvent
